RANSOD

αβχ Superoxide dismutase

MANUAL
RX MONZA

INTENDED USE SAFETY PRECAUTIONS AND WARNINGS

For the quantitative in vitro determination of Superoxide For in vitro diagnostic use only. Do not pipette by mouth.
dismutase in whole blood. This product is suitable for Exercise the normal precautions required for handling
manual use and on the Rx Monza. laboratory reagents.

Cat. No. Health and Safety data sheets are available on request.
SD 125 R1a. Mixed Substrate 5 x 20 ml
5 x 20 ml R1b. Buffer 1 x 105 ml The reagents must be used only for the purpose
R2. Xanthine Oxidase 3 x 10 ml intended by suitably qualified laboratory personnel,
CAL Standard 5 x 10 ml under appropriate laboratory conditions.

ASSAY PRINCIPLE STABILITY AND PREPARATION OF REAGENTS

The role of superoxide dismutase (SOD) is to accelerate the R1a. Mixed Substrate
•
dismutation of the toxic superoxide radical (02 ), produced Reconstitute the contents of one vial of Mixed
during oxidative energy processes, to hydrogen peroxide Substrate R1a with 20 ml of Buffer R1b. Stable for 10
and molecular oxygen. days when stored at +2 to +8°C.
This method employs xanthine and xanthine oxidase (XOD)
to generate superoxide radicals which react with R1b. Buffer
2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium Contents ready for use. Stable up to the expiry date
chloride (I.N.T.) to form a red formazan dye. The when stored at +2 to +8°C.
superoxide dismutase activity is then measured by the
degree of inhibition of this reaction. One unit of SOD is that R2. Xanthine Oxidase
which causes a 50% inhibition of the rate of reduction of Reconstitute one vial of Xanthine Oxidase R2 with
INT under the conditions of the assay. 10 ml of redistilled water. Stable for 2 weeks when
XOD
•
stored at +2 to +8°C.
Xanthine Uric acid + 02
•
CAL Standards
02 Reconstitute one vial of Standard (CAL) with 10 ml of
I.N.T. formazan dye redistilled water. Subsequent dilutions of this standard
should be prepared with Ransod sample diluent.
OR
•. •. +
SOD R1 = Mixed Substrate
02 + 02 + 2H 02 + H202 R2 = Xanthine Oxidase

SAMPLE PREPARATION It is recommended that all reagents and samples are

Use heparinized or EDTA whole blood samples. It is equilibrated to room temperature prior to use.
recommended that erythrocytes should be washed four
times with 0.9% NaCl solution. MATERIALS PROVIDED
Mixed Substrate
Centrifuge 0.5 ml of whole blood for 10 minutes at Buffer
3000 rpm and then aspirate off the plasma. Then wash Xanthine Oxidase
erythrocytes four times with 3 ml of 0.9% NaCl solution Standard
centrifuging for 10 minutes at 3000 rpm after each wash.
MATERIALS REQUIRED BUT NOT PROVIDED
The washed centrifuged erythrocytes should then be made Ransod Diluent (Cat. No. SD 124) (0.01 mol/l Phosphate
up to 2.0 ml with cold redistilled water, mixed and left to Buffer, pH 7.0)
stand at +4°C for 15 minutes. The lysate is diluted with 0.01 Ransod Control (Cat. No. SD 126)
mol/l Phosphate Buffer pH 7.0, so that the % inhibition falls
between 30% and 60%.

A 25 fold dilution of lysate is recommended for human

samples (Final dilution factor = 100) and a 50 fold dilution
for bovine samples (Final dilution factor = 200).

REAGENT COMPOSITION

Contents Initial Concentration of Solutions

R1a. Mixed Substrate

Xanthine 0.05 mmol/l
I.N.T. 0.025 mmol/l
R1b. Buffer
CAPS 40 mmol/l, pH 10.2
EDTA 0.94 mmol/l
R2. Xanthine Oxidase 80 U/l
CAL Standard See assigned value on Lot Specific Insert
MANUAL/ RX MONZA - RANSOD - SD 125 PAGE 2 OF 3

PROCEDURE CALCULATION
Select SOD in the Run Test screen and carry out a water A2 - A1
blank as instructed. = −A/min of standard or sample
Pipette into a cuvette: 3
Standard Standards Samples Control
S1 S2 TO S6 Sample diluent rate (S1 rate) = rate of uninhibited reaction
Ransod Sample Diluent 30 µl - - - = 100 %
Standard - 30 µl - -
Diluted Sample - - 30 µl -
Diluted Control - - - 30 µl All standard rates and diluted sample rates must be
Mixed substrate (R1) 1000 µl 1000 µl 1000 µl 1000 µl converted into percentages of the sample diluent rate, and
subtracted from 100 % to give a percentage inhibition.
Mix well and add :-
(−Astd/min x 100)
Xanthine Oxidase (R2) 150 µl 150 µl 150 µl 150 µl 100 - = % inhibition
(−AS1/min)
Mix well, insert the cuvette into the Rx Monza cell holder
and press read. (−Asample/min x 100)
100 - = % inhibition
CALIBRATION (−AS1/min)
Calibrate this assay using the standard supplied with the kit.
It is recommended that the following dilutions are made of Plot percentage inhibition for each standard against Log10
the Standard CAL (or S6) to produce a standard curve:- (standard conc. in SOD units/ml)

Standard Volume of Use percentage inhibition of sample to obtain units of SOD

Standard Soln Sample Diluent from standard curve.

S6 Undiluted Standard - SOD units/ml of whole blood =

S5 5 ml of S6 5 ml
S4 5 ml of S5 5 ml SOD units/ml from std curve x dilution factor
S3 5 ml of S4 5 ml
S2 3 ml of S3 6 ml Converting to SOD units/g Haemoglobin
SOD units /ml
S1 = Sample Diluent (0.01 mol Phosphate buffer pH 7.0) = SOD units/g Haemoglobin
All diluted standards are stable for 2 weeks at +2 to +8°C. g Haemoglobin/ml
A fresh calibration is required for each run.
ILLUSTRATION
FOR MANUAL USE (a) A bovine sample was diluted 1 in 300 times with Ransod
sample diluent. The diluted sample gave an inhibition of
Wavelength: 505 nm 33 %.
Cuvette: 1 cm path length From the standard curve:
Temperature: 37°C
Measurement: against air Number of SOD units in sample = 0.575

Pipette sample and R1 into a cuvette and mix well. Add R2,
mix and read initial absorbance A1 after 30 seconds and
start timer simultaneously read final absorbance A2 after 3
minutes.
(b) Converting to SOD units/ml of whole blood
0.575 x 300 = 172.5 SOD units/ml
(c) Converting to SOD units/g Haemoglobin

Sample haemoglobin value = 0.118 g/ml

172.5
(SOD units/ml)/(g Haemoglobin/ml) = = 1461.9
0.118

QUALITY CONTROL
Ransod Control is recommended for daily quality control. The
control should be assayed at least once a day. Values
obtained should fall within a specified range. If these values
fall outside the range and repetition excludes error, the
following steps should be taken:
1. Check instrument settings and light source.
2. Check cleanliness of all equipment in use.
3. Check water, contaminants i.e. bacterial growth may
contribute to inaccurate results.
4. Check reaction temperature.
5. Check expiry date of kit and contents.
6. Contact Randox Laboratories Customer Technical
Support, Northern Ireland (028) 94422413.

RANDOX Laboratories Ltd., 55 Diamond Road, Crumlin, Co. Antrim, United Kingdom, BT29 4QY

ΑΒΧ
Tel: +44 (0) 28 9442 2413 Fax: +44 (0) 28 9445 2912
Email: applications@randox.com Website: www.randox.com
MANUAL/ RX MONZA - RANSOD - SD 125 PAGE 3 OF 3

NORMAL RANGES
1102 - 1601 U/g Hb
164 - 240 U/ml

It is recommended that each laboratory establish its own

reference range to reflect the age, sex, diet and
geographical location of the population.

SPECIFIC PERFORMANCE CHARACTERISTICS

The following performance data were obtained using an Rx
Monza analyser in cuvette mode at 37ºC.

LINEARITY
Samples should be diluted to give an inhibition between
30 % and 60 % of the sample diluent rate (i.e. the
uninhibited reaction).

SENSITIVITY
The minimum detectable concentration of SOD less than
standards should be reported as <S1 standard value.

PRECISION

Within run precision

Level 1 Level 2
Mean 101.0 131.5
SD 4.88 4.30
CV(%) 4.64 3.58
n 20 20

Between run precision

Level 1 Level 2
Mean 101.0 131.5
SD 6.27 8.50
CV(%) 5.96 7.07
n 20 20

CORRELATION
The Randox method on the Rx Monza (Y) was compared to
the Rx Daytona (X) and the following linear regression
equation was obtained:

Y = 0.9417 X + 0.1004
and a correlation coefficient of 0.965

41 patient samples were analysed spanning the range 23 -

318U/ml.

REFERENCES
1. Woolliams JA, Wiener G, Anderson PH, McMurray
CH Research in Veterinary Science 1983, 34: 253-256.
2. Suttle NF, The Veterinary Record 1986, 119: 519-522.
3. Suttle NF, McMurray CH Research in Veterinary
Science 1983; 35: 47-52.
4. Arthur JR, Boyne R Life Sciences 1985 36: 1569-1575.

Revised 07 Oct 09 ck

RANDOX Laboratories Ltd., 55 Diamond Road, Crumlin, Co. Antrim, United Kingdom, BT29 4QY

ΑΒΧ
Tel: +44 (0) 28 9442 2413 Fax: +44 (0) 28 9445 2912
Email: applications@randox.com Website: www.randox.com